The Role of Cystatins in Cells of the Immune System

FEBS Letters 580 (2006) 6295–6301

Minireview The role of cystatins in cells of the immune system

Natasˇa Kopitar-Jerala* Department of Biochemistry and Molecular Biology, Jozˇef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia

Received 16 August 2006; revised 22 October 2006; accepted 24 October 2006

Available online 3 November 2006

Edited by Masayuki Miyasaka

The cystatins constitute a large group of evolutionary Abstract The cystatins constitute a large group of evolutionary related proteins with diverse biological activities. Initially, they related proteins acting as protease inhibitors of papain-like were characterized as inhibitors of lysosomal cysteine proteases cysteine proteases belonging to enzyme family C1 (see the – cathepsins. Cathepsins are involved in processing and presenta- MEROPS database at http://merops.sanger.ac.uk), such as tion of antigens, as well as several pathological conditions such cathepsins B, H, L, and S and legumain-related proteases of as inflammation and cancer. Recently, alternative functions of the family C13 [10]. Type 1 cystatins, stefins (A and B), are cystatins have been proposed: they also induce tumour necrosis polypeptides of 98 amino acid residues which possess neither factor and interleukin 10 synthesis and stimulate nitric oxide disulfide bonds nor carbohydrate side chains and are located production. The aim of the present review was the analysis of mainly intracellularly. Type 2 cystatins C, D, E/M, F, S, SN, data on cystatins from NCBI GEO database and the literature, and SA are characterized by two conserved disulfide bridges, and obtained in microarray and serial analysis of gene expres- a larger size (120 residues) and the presence of a signal sion (SAGE) experiments. The expression of cystatins A, B, C, and F in macrophages, dendritic cells and natural killer cells peptide for extracellular targeting [11]. Type 3 cystatins, the of the immune system, during differentiation and activation is kininogens, are large (60–120 kDa) multifunctional plasma discussed. proteins, containing three type 2 cystatin-like domains con- Ó 2006 Federation of European Biochemical Societies. Published taining a total of eight disulfide bridges. Although types 1 by Elsevier B.V. All rights reserved. and 2 cystatins display considerable differences in amino acid sequence, their tertiary structures are conserved and exhibit a Keywords: Cystatin; Cathepsin; Macrophages; Dendritic cells; ‘cystatin fold’ that is formed by five stranded anti-parallel b- Natural killer cells pleated sheet wrapped around a five-turn a-helix [12,13]. The structure of human cystatin C in its dimeric form also shows that each one of the two domains in the dimer adopt the typ- ical monomeric ‘cystatin fold’ [14]. Some type 2 cystatins (C, E/M, and F) are also able to inhibit mammalian legumain, 1. Introduction an asparaginyl endopeptidase (AEP), using a binding site distinct from the family C1 interaction site [15]. AEP has been Cysteine cathepsins are long known to be responsible for shown to be involved in class II major histocompatibility com- protein degradation in lysosomes. Recent studies show that plex (MHC) restricted antigen presentation [16]. they are also involved in a number of other important cellular The present review focuses mostly on the expression of two processes such as antigen presentation [1], apoptosis, protein type 1 cystatins: stefins (cystatins) A and B and two type 2 cyst- processing [2], as well as several pathologies such as cancer atins, cystatins C and F, in cells of the immune system upon progression [3], inflammation [4] and neurodegeneration [5]. differentiation and activation. Two recently developed technol- The role of lysosomal cathepsins in antigen presentation has ogies, oligonucleotide or cDNA microarrays and serial analy- been reviewed recently [6]. Proteinase activity in these pro- sis of gene expression (SAGE), allow the determination of the cesses is highly regulated at the level of protease expression, expression patterns of thousands of genes simultaneously by regulation of zymogen activation and by expression of [17,18]. The gene expression omnibus (GEO) at the National endogenous inhibitors. Natural inhibitors that inhibit cysteine Center for Biotechnology Information (NCBI) is a large com- cathepsins include cystatins, thyrophins and also some of the pendium of gene expression data, addressing a wide range of serpins [7–9]. biological issues across many organisms [19]. The aim of the present review is the identification of some of the most interesting questions regarding cystatins in cells of the immune system on the basis of recent data collected in the NCBI GEO and the *Fax: +386 1 477 39 84. literature. E-mail address: [email protected]

Abbreviations: BCR, B cell receptor; DC, dendritic cells; FDC, follicular dendritic cells; GC, germinal centres; SAGE, serial analysis of 2. Steﬁn A (cystatin A) in follicular dendritic cells (FDC) gene expression; MHC, major histocompatibility complex; PKC, protein kinase C; TNF-a, tumour necrosis factor alpha; IL-4, interleukin-4; IL-10, interleukin-10; IFN-c, interferon-gamma; NK, natural Steﬁn A (cystatin A) has been isolated from epidermis, poly- killer cells morphonuclear granulocytes, liver, and spleen [20–23]. SAGE

0014-5793/$32.00 Ó 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.10.055 6296 N. Kopitar-Jerala / FEBS Letters 580 (2006) 6295–6301 studies showed that LPS stimulation decreased its synthesis in activity on neural stem cells [47]. Cystatin C N-glycosylation monocytes [24]. Stefin A was also found in the follicular den- was necessary to induce neural stem cell proliferation. The pro- dritic cells (FDC) in germinal centres (GC) of human tonsils tease inhibitory domain of cystatin C was not directly involved [25]. In contrast to antigen-presenting cells that present anti- in the process [47]. The unexpected consequences of cystatin gens to T cells, FDC do not internalize, process and present C deficiency on the spread of metastasis and atherosclerosis antigens in the context of major histocompatibility complex could also be a consequence of alternative functions of cystatin class II (MHC II), but present intact antigen on their cell C, possibly as growth factor cofactor. surface [26,27]. GC form in lymphoid follicles of secondary Dendritic cells (DC) are the professional antigen presenting lymphoid organs and provide an essential microenvironment cells of the immune system. They are defined functionally by for T cell-dependent humoral immune responses [28,29]. their ability to take up antigens such as microorganisms, pro- Within GC, antigen-specific B cells efficiently undergo clonal cess them into short antigenic peptides, load the peptides onto expansion, isotype switching, somatic mutation, and affinity major histocompatibility complex (MHC) molecules and then maturation leading to the generation of plasma and memory present the resulting complexes at the cell surface. Immature cells [30–32]. Only B-cells with the highest affinity B cell recep- DC are located in the periphery of the body and they take tor (BCR) bind to intact antigens on the surface of FDC and up and process antigens. Activated DC lose their capacity to receive survival signals from the FDC whereas low affinity capture and process antigens. Instead they migrate to the sec- BCR B cells and self-reactive B cell clones are eliminated by ondary lymphoid organs and present antigen to T cells [48]. apoptosis [31]. Apoptosis in GC B-cells is mainly induced via Self peptides derived from secretory membrane proteins that the death receptor pathway, by the rapid activation of cas- are synthesized by the antigen-presenting cells themselves bind pase-8 at the level of CD95 death-inducing signalling complex to MHC class II molecules tightly, but normally do not acti- (DISC) [33–35]. GC B cell apoptosis is dependent not only on vate T cells. Cystatin C peptide (amino acids 40–55) has been caspases but also endonucleases [36] and cathepsins [37]. Re- found as one of such self peptides bound to MHC class II mol- cently, van Nierop et al. showed that apoptosis in human ecules, indicating that it is endocytosed and cleaved with the GC B-cells involved lysosomal destabilization, which was con- antigenic material and then bind to MHC class II molecules trolled by caspase-8 activity. CD40 ligation provided resistance [49]. Hashimoto et al. [50] observed that upon DC maturation to lysosomal destabilization, as well as binding of high-affinity cystatin C transcripts were significantly downregulated (http:// B cells to FDC prevented lysosomal leakage and apoptosis in bloodsage.gi.k.u-tokyo.ac.jp/). The SAGE results of Hashim- GC B-cells [38]. Van Nierop et al. speculated that besides cas- oto et al. were confirmed at the protein level by Zavasnik-Ber- pase-8 inhibition there was an additional mechanism to pre- gant et al. who observed a large increase in intracellular vent lysosomal instability in adhering GC B-lymphocytes: cystatin C during the differentiation of monocytes to immature stefin A, which is expressed at high levels in FDC may play DC [51], Upon DC maturation, intracellular cystatin C levels a role in the prevention of apoptosis, as previously proposed decreased and following prolonged incubation of mature DC by van Eijk et al. [37,39]. in the presence of TNF-a, cystatin C was secreted from DC. It has been proposed that cystatin C plays a pivotal role in the control of cleavage and removal of the MHC class II 3. Cystatins B and C in dendritic cells and macrophages invariant chain (Ii) by regulating the activity of cathepsin S, and hence in the formation of MHC class II-peptide complexes Cystatin C is the most potent inhibitor of cysteine proteases [52]. The work of El Sukkari et al. on DC isolated from cyst- such as cathepsins B, H, L, and S, with apparent inhibition atin C-deficient mice showed that cystatin C is neither neces- constants even below the nanomolar range [40]. Mature cysta- sary nor sufficient to control MHC class II expression and tin C is synthesized as a preprotein with a 26 residue signal antigen presentation in DC and that its expression differs be- peptide. Cystatin C is ubiquitously expressed in all tissues tween different DC subsets [53]. The absence of cystatin C and cell types, although mRNA levels vary several-fold be- did not affect the expression, subcellular distribution, or for- tween the tissues [41,42]. Oligomerization of cystatin C leads mation of peptide-loaded MHC class II complexes in any of to amyloid deposits in brain arteries at advanced age but this the DC types, nor the efficiency of presentation of exogenous pathological process is greatly accelerated in the mutant form antigens [53]. Recent work by Kitamura et al. showed that of cystatin C, responsible for hereditary cystatin C amyloid interleukin-6 (IL-6)-mediated signalling increased cathepsin S angiopathy (HCCAA) [43]. Extracellular monomeric cystatin activity, significantly reduced cystatin C expression and re- C was found to be internalized by Chinese hamster ovary duced the H2-DM and MHC class II ab dimer levels in DC (CHO) cells and trafficked into lysosomes where it dimerized [54]. Overexpression of cystatin C in DC on the other hand sig- [44]. Cystatin C-deficient mice have essentially a normal phe- nificantly suppressed IL-6-mediated enhancement of cathepsin notype. Cystatin C-deficient mice showed significantly reduced S activity and reduction of MHC class II ab dimer, Ii, and H2- growth of melanoma lung metastases when compared to wild- DM levels in DC. The authors concluded that the IL-6-medi- type mice [45]. The reason for reduced growth of melanoma ated alteration of the balance between cystatin C and cathepsin lung metastases in cystatin C-deficient mice is unknown, but S levels is important for the status of MHC class II ab dimer, could be a consequence of an early proteolytic event by a cys- Ii, and H2-DM levels in DC. At least in the system described, teine proteinase during the first hours after administration of cystatin C may regulate cathepsin S activity in immature and melanoma cells [45]. Cystatin C-deficient mice also showed mature DC [54]. Murine spleen contains three major endo- that cystatin C has a protective role in atherogenesis since cyst- genous populations of DC. They are referred to as the CD8+ atin C-deficiency promotes atherosclerosis [46]. It was shown CD4À, CD8À CD4+, and CD8À CD4À subsets [55,56]. CD8À that a glycosylated form of cystatin C is a necessary cofactor DC are distinct from CD8+ DC on the basis of a number of for fibroblast growth factor 2 (FGF-2) induced mitogenic criteria and primarily direct a Th2 response by activating T N. Kopitar-Jerala / FEBS Letters 580 (2006) 6295–6301 6297 cells to secrete cytokines such as interleukin-4 (IL-4) [57,58]. tion of a cysteine proteinase activity since E-64 did not induce CD8+ DC produce IL-12 upon stimulation and induce a Th1 any increase in NO. Increased NO was due to increased induc- response [59]. Affymetrix microarray gene analysis was used ible NO synthase protein synthesis. Further studies of Verdot to determine gene expression patterns among murine DC sub- et al. suggested that chicken cystatin stimulated the release of sets: CD8+ CD4À and CD8À CD4À cells were analyzed directly TNF-a and interleukin-10 (IL-10) by IFN-c-activated murine after sorting and after 2 h cultivation (NCBI GEO GDS352). peritoneal macrophages [74]. This observation could be of bio- Cystatin C was upregulated in cultured CD8+DC (NCBI logical importance as cystatin concentrations necessary to GEO GMS4772, GMS4773), an observation that is in agree- upregulate TNF-a, IL-10 and NO synthesis are in the physio- ment with the study of El-Sukkari et al. [53]. DC also have logical range, as found in human body fluids. The cystatin C- the capacity to take up, process and present exogenous anti- mediated release of TNF-a is probably responsible for the gens in association with MHC class I molecules and this path- increase in NO production by IFN-c-activated murine perito- way is termed cross-presentation [60–62]. CD8+ DC have been neal macrophages. The findings by Verdot et al. [74] point at shown to be the principal DC subset involved in priming MHC a new relationship between cystatins, cytokines, inflammation class I-restricted cytotoxic T cell immunity [63]. Since cystatin and immune responses. C is expressed predominately in CD8+DCs, it is possible that it In vitro experiments in cell culture models described above has a role in this process. were confirmed by experiments in Leishmania donovani in- Stefin B (cystatin B) is a type 1 cystatin that is distributed fected mice [75]. L. donovani, the etiological agent for the rather uniformly among different tissues. In vitro stefin B binds severe visceral form of leishmaniasis, multiplies in the phago- tightly to cathepsins H, L, and S, and less tightly to cathepsin lysosomes of macrophages of the infected host. Treatment of B [9]. Mutations in the gene encoding stefin B are responsible L. donovani-infected murine peritoneal macrophages with a for the primary defect in Unverricht-Lundborg disease combination of chicken cystatin and IFN-c induced increased (EPM1) [64–66]. Stefin B-deficient mice display a phenotype production of NO and did overcome the inhibition of NO that is similar to the human disease with progressive ataxia synthesis driven by L. donovani parasites. Mice treated with and myoclonic seizures [67]. The mice exhibit apoptosis of cer- chicken cystatin and IFN-c showed reduced splenomegaly, a ebellar granullar cells and show increased expression of apop- lowered parasite burden in the spleen and increased produc- tosis and glial activation genes [68]. It was shown that removal tion of NO [75]. The infected mice treated with chicken cysta- of cathepsin B from cystatin B-deficient mice greatly reduced tin and IFN-c were cured by the induction of NO that killed neuronal apoptosis, but did not rescue animals from ataxia the parasite and the switched CD4+ T cell-mediated immune and seizure [69]. Thymocytes from stefin B-deficient mice ex- responses from disease-promoting Th2 cells to the protective erted a markedly increased response when they were exposed Th1 response shown by the increased production of IL-12 to staurosporin, a protein kinase C (PKC) inhibitor compared and decreased production of IL-4. to thymocytes from wild-type mice [70]. We tested the possibil- ity that stefin B interacts with the receptor for activated PKC (RACK-1) in thymocytes and in this way interferes with PKC 4. Cystatin F in NK cells signaling in the cells, but the interaction of RACK-1 with stefin B was not confirmed. Preincubation of cells with E-64d did Cystatin F is expressed in a variety of tissues. Expression is not prevent apoptosis, indicating that staurosporin induces particularly high in the cells and tissues of the immune system: apoptosis in a cathepsin-independent and caspase-dependent thymus and spleen, monocytes, DC, T-cells and NK cells [76]. manner. Brannvallet et al. reported that stefin B is localized Mature cystatin F is composed of 126 amino acid residues. It is mainly in the nucleus of neural steam cells and in neurons, synthesized as a preprotein with a 19 residue signal peptide and while in glia cells it is also in the cytoplasm and in the lyso- possesses a unique extension of six amino acids at its N-termi- somes [71]. Hashimoto et al. showed that gene transcripts nus. In addition to the two disulfide bridges common to all of stefin B were significantly increased upon differentiation of type 2 cystatins, mature cystatin F has two cysteine residues monocytes into macrophages [72]. However, upregulation of that, form an interinolecular disulfide bridge, as revealed in the expression of the inhibitor upon differentiation of macro- the crystal structure of the cystatin F dimer [77]. Cappello phages does not result in co-localization or interaction with et al. observred that in U937 cells cystatin F was secreted as cathepsins L, S or B (Kopitar-Jerala, unpublished observa- a disulfide bridge-linked dimer [78]. Cystatin F dimer is inac- tions). The SAGE studies showed that treatment with LPS tive as an inhibitor of papain like cathepsins and can be causes upregulation of stefin B expression in human mono- activated by chemical reduction [79]. As compared to other cytes, whereas cystatin C is not affected, indicating a possible cystatins, the protein exhibits a distinct inhibitory profile. It role of stefin B in innate immune response to bacterial infec- binds tightly to cathepsins F, K, L, and V, less tightly to tions [24]. cathepsins S and H, and does not inhibit cathepsins B, C, Activated macrophages acquire antimicrobial activities and X [79]. Cystatin F can inhibit AEP, but with lower affinity involving reactive oxygen species and reactive nitrogen metab- as cystatin E/M and C [80]. The recently elucidated crystal olites. Chicken cystatin, cystatin C, and stefin B have been structure of cystatin F revealed that two N-linked glycosyla- implicated in nitric oxide (NO) production by interferon-c- tion sites of cystatin F may modulate its inhibitory properties, activated mouse peritoneal macrophages [73]. Mouse perito- in particular its reduced affinity towards AEP as compared to neal macrophages activated with interferon-gamma (IFN-c) other cystatins [77]. and then stimulated with IFN-c plus chicken cystatin gener- Cystatin F has also been shown to be strongly upregulated ated increased amounts of NO in comparison with macro- in LPS-stimulated monocyte-derived DC [50]. In the U937 phages only activated with IFN-c [73]. The biological effect premyeloid cell line, both differentiation towards a granulo- of cystatins as NO synergistic inducers is not related to inhibi- cytic pathway by all-trans-retinoic acid (ATRA) or towards 6298 N. Kopitar-Jerala / FEBS Letters 580 (2006) 6295–6301

Fig. 1. Steﬁn A (cystatin A) is expressed at high levels in FDC and may play a role in the prevention of apoptosis in GC B cells as proposed by van Eijk et al. [37,39]. Several signalling molecules are involved in FDC-GC B cell contacts: intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) enhance cell-cell contact; B-cell-activating factor of the tumour necrosis factor family (BAFF/BLys) prevents apoptosis of GC B cells and interleukin-15 (IL-15) stimulate GC B-cell proliferation [28].

a monocytic pathway by stimulation with phorbol ester (TPA) cytes and the majority of human NK cells are CD56dim, resulted in marked downregulation of cystatin F expression whereas a minority are CD56bright and CD16dim/neg [84]. The [81]. function of CD56bright NK cells is different from that of In U937 cells, cystatin F has been found to be secreted and CD56dim NK cells. CD56bright NK cells can produce cytokines localized intracellularly in lysosome-like granules [81]. Cap- more abundantly, consistent with their functional role as an in- pello et al. found it in lysosomes in transfected HeLa cells, nate immunoregulator [86]. In contrast, CD56dimCD16+ NK but not in U937 cells. Sorting of cystatin F to lysosomes was cells seem to be skewed toward homing to inflammation sites greatly enhanced when its C-terminal end was extended by and promoting immune responses, in addition to induction few amino acids [78]. The authors concluded that under partic- of cytotoxicity [86]. ular conditions, cystatin F can be sorted to the endocytic path- Hanna and coworkers reported that cystatin F tran- way, but its unusual inhibitory function is mainly performed scripts were more abundant in CD56dimCD16+ NK cells and extracellularly and probably controlled through dimerization in vitro activated CD56+CD16+ NK cells than in [78]. Langerholc et al. showed that in U937 cells, cystatin F CD56brightCD16À NK cells [87] (NCBI GEO GSM26200-5). was co-localized with lysosomal markers LAMP-2 and CD68 Although the microarray data do not give us any information and when subcellular localization of cystatin F was compared about inhibitory activity of cystatin F and have to be inter- to that of cathepsins, cystatin F was found to be co-localized preted with caution, it is tempting to speculate that cystatin with cathepsins X and H, but not with cathepsins L, B, C F plays a specific role in the function of NK (CD56dimCD16+) [79]. Further investigations on cystatin F localization, possibly cells. in other cells and in cystatin F-deficient mice, will be necessary to elucidate its biological function (see Fig. 1). Gene expression analysis of human NK cells and CD8+ T 5. Conclusions lymphocytes revealed that transcripts of cystatin F were significantly upregulated in NK cells when compared to CD8+T Several structural and kinetic studies have given us insight lymphocytes (http://bloodsage.gi.k.u-tokvo.ac.ip/) [82]. into interactions of cystatins and cysteine cathepsins in vitro NK cells are innate immune lymphocytes that mediate two but in vivo very few interactions have been found. The present major functions: recognition and lysis of cancer cells and review aims to identify the most interesting questions rather virus-infected cells and production of immunoregulatory cyto- than providing the definitive answers. The central question kines [83,84]. The activation of NK cells is controlled by com- remains what the targets of cystatins are that are differentially plex interactions between activating and inhibitory receptor regulated in cells of the immune system. Is it possible that cyst- signals and can be modulated by cytokines [85]. 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